A detailed 3D MRI brain atlas of the African lungfish Protopterus annectens

The study of the brain by magnetic resonance imaging (MRI) in evolutionary analyses is still in its incipient stage, however, it is particularly useful as it allows us to analyze detailed anatomical images and compare brains of rare or otherwise inaccessible species, evolutionarily contextualizing possible differences, while at the same time being non-invasive. A good example is the lungfishes, sarcopterygians that are the closest living relatives of tetrapods and thus have an interesting phylogenetic position in the evolutionary conquest of the terrestrial environment. In the present study, we have developed a three-dimensional representation of the brain of the lungfish Protopterus annectens together with a rostrocaudal anatomical atlas. This methodological approach provides a clear delineation of the major brain subdivisions of this model and allows to measure both brain and ventricular volumes. Our results confirm that lungfish show neuroanatomical patterns reminiscent of those of extant basal sarcopterygians, with an evaginated telencephalon, and distinctive characters like a small optic tectum. These and additional characters uncover lungfish as a remarkable model to understand the origins of tetrapod diversity, indicating that their brain may contain significant clues to the characters of the brain of ancestral tetrapods.

availability of rare models in the laboratory, which may be accessible to the scientific community through the large number of resources that have been emerging in recent years with these analyses.In fact, apart from its main clinical use in humans, or even in non-humans, its use in research is becoming more widespread in the last years, as evidenced by the project Digital Fish Library for the case of fishes (http:// www.digit alfis hlibr ary.org/).Recently, studies of the cranial endocast of sarcopterygian fishes have been developed using non-invasive technology, either with fossil or living species [43][44][45] .However, these approaches lack a detailed neuroanatomical interpretation.In this study, we have identified and segmented for the first time the main brain regions of a representative species of lungfish, Protopterus annectens, using MRI approach.Thus, we have developed a threedimensional (3D) model of the brain showing its main regions and the ventricular system, as well as providing volumetric measures of the brain and the ventricular system.

Results and discussion
The brain of the lungfish P. annectens has been divided in the main regions shared by most vertebrates: olfactory bulb, telencephalon, hypothalamus, diencephalon, mesencephalon, cerebellum, and rhombencephalon.In addition, other subdivisions, such as the accessory olfactory bulb, pallium, subpallium, and preoptic area within the telencephalon, the hypophysis, and the alar-basal boundary, have also been identified and analyzed in this study.The segmentation of the different regions, together with the ventricular system, was performed manually using previous neuroanatomical studies as reference [23][24][25][26][27][28]31,35,36 (Fig. 2; see list of colors and abbreviations in Table 1). Based onthese segmentations, a three-dimensional model was built (Figs. 3, 4 and 5).The data of the variables considered (body length and weight, brain and ventricular volumes, and the relation between the latter two) are detailed in Table 2, while the correlation analyses of them appear in Table 3.The correlation observed between the variables body length, body weight and brain volume is high, as well as the correlation between brain and ventricular volumes.In the case of the latter, the correlation with the rest of the variables is considerable, but lower, most probably due to the sometimes-difficult interpretation of the extent of the ventricles by MRI, particularly in zones like the third or the fourth ventricle, where the tela choroidea is not clearly distinguishable with this technique.
This neuroanatomical technique has been widely used in humans, mostly with a clinical purpose 46,47 , but, like in our case, it has also been leveraged to study the brains of several models of all groups of vertebrates, i.e. mammals 48,49 , birds [50][51][52] , reptiles [53][54][55] , amphibians 56 , and fish [57][58][59][60][61][62] .Lungfishes are a key model in evolutionary analysis due to their phylogenetic position, since they constitute the only bony fish with an evaginated brain more closely related to amphibians than to other fishes.Classical histological [18][19][20] and recent genoarchitectonic studies [23][24][25][26][27] have suggested that these neuroanatomical features may be comparable to those of the ancestors of amniotes.Therefore, the neuroanatomic characteristics that we are going to detail below could be discussed in this context.

Olfactory bulb
Located in the anterodorsal telencephalon, and sessile on the rostral part of the telencephalic hemispheres, the secondarily evaginated olfactory bulbs of Protopterus present a laminar structure and large ventricles (Fig. 2A,B).In addition, in the ventrolateral part appears the accessory olfactory bulb in caudal levels 26 (Figs.3B, 5A).In particular, regarding the vomeronasal system, it was shown that, in terms of processing this information, lungfishes  share a basic organization with tetrapods 26 , which allowed us to suggest that these features were already present in the last common ancestor of lungfishes and tetrapods.

Pallium and subpallium
The telencephalic roof houses the pallium, while the subpallium (septum medially and striatum laterally) is located in the floor (Figs.3B, 4B,D, 5A).Although there is no external evidence of the pallial-subpallial boundaries, internally they are revealed by changes in cellular density and thickness of the cell plate.However, the divisions of the different pallial fields, i.e., medial, dorsal, lateral and ventral, and the subpallial nuclei were not clearly distinguishable by MRI (Fig. 2A-E).The telencephalon of this model exhibits one of the unique characteristics of sarcopterygian fishes, which is its development by evagination, in contrast to actinopterygian fishes whose telencephalon develops by eversion.This feature, together with its neurochemistry and regionalization (the organization of the pallium into at least four domains 24 , the existence of subpallial regions comparable to that of tetrapod 29,33 , as well as the conserved neurochemical systems 24,34,36 ), has made the telencephalon of this model very similar in appearance to that of amphibians, and in particular to anurans 27,37 .From an evolutionary point of view this is of great interest as it supports the possible proximity of these model to the ancestor of amniotes and its position as a key model in these evolutionary studies.

Preoptic area
This caudal subpallial region develops from the nonevaginated part of the telencephalon 63 .It is situated ventrally, in the territory of the preoptic recess of the third ventricle (Figs.2E, 4D), with which most of its cells send cerebrospinal fluid contacts.The neurochemistry, genoarchitecture and organization of this region has been shown to be highly conserved with respect to that described in tetrapod sarcopterygians 25,32,33 .

Hypothalamus
Following the prosomeric model, the alar-basal boundary courses along the rostrocaudal extension of the brain, but folds almost 90º in the diencephalic region, dividing the hypothalamus in an alar portion (rostral in classical view, but dorsal in the updated prosomeric view) and a basal portion (caudal in classical view, but ventral in prosomeric view) (Figs.3B, 4D, 5A).The alar portion houses the paraventricular (with the neurosecretory nuclei) and the subparaventricular regions, the latter of which contains the suprachiasmatic nucleus (Fig. 2F), while the basal portion is composed of the tuberal and the mamillary areas (Fig. 2G-I).The interpretation of the hypothalamus of this model following the subdivisions proposed in the prosomeric model is recent, and it has been based on the expression of main morphogens of this basal territory such as Nkx2.1 and Isl1 33 and the neurochemistry of the hypothalamus itself, as reflected in the catecholaminergic populations 31 , which are highly conserved in evolution.In addition, also in the caudal part of the basal portion is situated the prominent hypophysis (Figs.2J, 3B, 4D, 5A).

Diencephalon
This region can be segmented in 3 prosomeres that possess alar and basal portions: p3, which includes the prethalamic eminence and prethalamus in its alar portion and the posterior tubercle in the basal part; p2, whose www.nature.com/scientificreports/alar portion is composed of the thalamus and the epithalamus, including the habenula and the epiphysis; and p1, with the pretectum in its alar part, whereas the basal portions of p1 and p2 are smaller (Figs.2F-H, 3B, 4B, 5A).These MRI-evidenced subdivisions have been precisely delineated on the basis of the expression of Pax6 transcription factors in combination with Isl1 expressed in the prethalamus 36 , as well as its boundary with the calcium-binding protein-rich thalamus 34 .The combination of both types of approaches has allowed the interpretation of MRI studies to have much greater depth of analysis based on this prior information.

Mesencephalon
It is segmented in an alar portion, which houses the optic tectum and the torus semicircularis, and the basal portion, where the mesencephalic tegmentum is situated housing the oculomotor nucleus (Figs.2I-K, 3B, 4B, 5A).These MRI results in the case of the midbrain are particularly interesting because they allow us to demonstrate its relative size, already described but without exact measurements, since the midbrain of lungfish represents a very small portion of the brain, with a bilaterally poorly developed optic tectum 18,19 , which may be a reflection of their ecological adaptations 22 .Although neurochemical populations conserved in other vertebrates have been described, as well as evolutionary particularities specific to lungfish, such as the superficial mesencephalic nucleus, located in a subpial position in the lateroventral margin of the alar midbrain 27 , the definition in this case is scarce.

Rhombencephalon
It is the most caudal region of the brain, divided into rhombomeres containing alar and basal portions (Figs.2J-N, 3B, 4B,D, 5A).There have been described eight rhombomeres, plus r0 or the isthmic region in the most rostral part, which houses the trochlear nucleus.All the motor nuclei of the cranial nerves, except for the oculomotor nucleus, are situated in the basal rhombencephalon.The basal portion also houses the reticular formation, with important components like the serotonergic raphe column in the medial line.In turn, the alar portion harbors the noradrenergic locus coeruleus, the cholinergic laterodorsal tegmental nucleus, the acoustic-vestibular nuclei and the nucleus of the solitary tract.Caudal to the obex, in the most caudal part of the rhombencephalon, begins the rostral portion of the spinal cord (Figs.2O, 3B, 4B,D, 5A).

Cerebellum
Within the rhombencephalon and poorly developed, this region expands from the alar part of r1 and folds caudally over the rostral fourth ventricle (Figs.2J-L, 3B, 4B, 5A).In this region, which had already been defined since classical studies [14][15][16] and more recently on the basis of Pax6 expression in its granule cells 36 , again, MRI analysis is very useful to identify it, as it is clearly observed and can be confidently defined rostrocaudally.It also opens the door to volumetric comparative studies.

Ventricular system and volumetric analysis
The MRI technology makes it possible to analyze accurately the ventricular system in a way that is unfeasible using histological techniques, that is, keeping it unaltered by possible deformations or contractions that are common when manipulating the tissue.Thus, we were able to develop a 3D representation of the entire system of cavities that greatly improves the understanding of its organization.In the telencephalon, two large lateral ventricles comprise its dorsoventral extent, from the pallium to the subpallium, reaching rostrally the olfactory bulbs and caudally joining into one cavity at levels corresponding to the caudal extent of the pallium (Figs.2B-D, 3D,E, 4C,E, 5B).This union results in the third ventricle (Figs.2E-H, 3D,E, 5B), which occupies the diencephalon.From its ventral part, the preoptic recess develops rostrally (Figs.2E, 3D, 4E) and the hypothalamic ventricle caudally (Figs.2G,H, 3D,E, 4C,E, 5B), the latter forming two lateral recesses in its caudalmost part (Fig. 2I).
In turn, in its dorsal part stands the epithalamic recess between the epiphysis and the habenulae (Fig. 2E,F).The third ventricle is then succeeded by the mesencephalic ventricle (Figs.2I, 3D, 4C,E, 5B), and this one leads caudally to the cerebellar ventricle, which forms two lateral extensions in its rostral part (Figs.2J,K, 3D, 4C,E, 5B).Finally, the rest of the rhombencephalon is occupied by the fourth ventricle (Figs.2L-N, 3D, 4B,C,E, 5B), which is opened dorsally and caudally closes itself to form the central canal that runs through the center of the spinal cord (Figs.2O, 3D, 4C,E, 5B).Therefore, MRI analysis of the ventricles has been of great interest and help, as it has allowed us to define these structures in lungfish with great precision for the first time.In addition, we have been able to perform a comparative volumetric study of these structures in relation to brain size (Tables 2 and 3).In relative size, the lungfish brain is large; however, the evolutionary interpretation of the cognitive consequences of having a large brain is not always obvious.Some authors have related the increase in cognitive capacities to an increase in the number of neurons, independently of volume 64 , while others consider that volume is an appropriate parameter since it is determined by the number of neurons 56 .In any case, regardless of the relationship with cognitive capacity, we consider that volumetric analyses by MRI are of great interest from an evolutionary point of view, not only in terms of total volume, but also in a partial way, between areas and specific territories.As discussed, in the case of lungfish, it would be very interesting to relate the relative size of the optic tectum or cerebellum to that of other models with different ecological characteristics and phylogenetic positions.

Conclusions
The MRI analysis of the brain of the lungfish Protopterus annectens provides valuable information, unattainable by histological methods.On the one hand, it has enabled us to measure brain and ventricular volumes, data that can be contrasted with those measures in other vertebrates in order to develop comparative neuroanatomical studies.On the other hand, we were able to use the MRI data to develop the first 3D brain atlas of a lungfish, granting a better comprehension of the neuroanatomy of these sarcopterygian fishes.

Animals
In this study seven young adult specimens of Protopterus annectens (total length 19.5-34 cm) were used.All the experiments described here were performed according to the ARRIVE guidelines and the regulations

Animal processing
All animals were profoundly anesthetized by immersion in 0.2% tricaine methanesulfonate solution (MS222, pH 7.4; Sigma-Aldrich Merck KGaA, Darmstadt, Germany) and perfused transcardially with 200 ml of 4% paraformaldehyde in 0.1 M phosphate buffer (PB, pH 7.4).The brain and the rostral spinal cord were extracted and stored firstly in the same fixative solution for 3 h at 4 °C and then in a solution of 30% sucrose in PB overnight at 4 °C.
The system consists of a 1 Tesla permanent magnet with a gradient coil that provides a gradient strength of 450 mT/m and a solenoid RF coil.Three-dimensional T2 coronal weighted images were acquired by using a rapid acquisition with relaxation enhancement (RARE) technique, with predefined parameters

Histological procedure
The other brain was processed for Nissl staining.In this way, it was introduced in a solution of 20% gelatin with 30% sucrose in PB, resulting in a block that was stored overnight in a 3.7% formaldehyde solution at 4 °C.The block was cut on a freezing microtome in 40 µm thick transverse slices, which were collected and rinsed in cold PB.The sections were then mounted on glass slides and stained with cresyl violet, and finally covered with Entellan ® (Sigma-Aldrich Merck KGaA).Photographs were taken with an Olympus DP74 camera coupled on an Olympus BX51 microscope (Olympus, Tokyo, Japan) and adjusted with Photoshop (Adobe Systems, San Jose, CA).The nomenclature used in the present study is the same that in previous studies of the brain of lungfish 25,26,[28][29][30][31][32][33][34][35][36][37] . Vol

Table 1 .
List of the segmented brain regions and associated colors and abbreviations.

Table 2 .
(A) Data of the variables measured and (B) chart of the variables brain and ventricular volumes.

Table 3 .
Correlation analysis of the variables length and weight of the brain, brain volume, and ventricular volume.